We wish to gain insight into the immune correlates of HIV protection by investigating potential mechanisms of HIV-1 resistance among high-risk, multiply exposed, seronegative (ES) individuals. Employing standardized assays and incorporating relevant control groups for comparative analyses, we found that most persons in the Seattle ES cohort lack HIV-1-specific IFN-gamma-secreting T cell responses. Moreover, their levels of CD4+ T cell infectivity with either R5- or X4-dependent viruses are similar to those in the lower risk control population. However, clear individual exceptions exist, and the fate of some ES over time has become quite intriguing. Some have seroconverted with viruses distinct from their known long-term infected sexual partners. Others remain seronegative but upon careful examination bear extremely low copy numbers of HIV-1 DNA. These results suggest that rare members within this cohort may have an unusual capacity to control HIV-1 infection that is distinct from CCR5 coreceptor impairment. We propose 3 specific aims to test the overall hypothesis that some long-term, multiply exposed seronegative persons have relative resistance to HIV infection that is maintained by their immune response. In Aim 1, we will ascertain and compare the frequency and reproducibility of HIV-1-specific IL-2- and IFN-gamma-secreting T cell responses in ES among different sexually-exposed risk groups. In Aim 2, we will assess the complete repertoire of anti-viral cellular responses in ES using multiparameter flow cytometry and array technology. In Aim 3, we will determine the contribution of HIV-1-specific T cell responses in ES who have unusual control of HIV-1 infection, either maintaining seronegativity with very low HIV-1 levels or following late seroconversion. We will conduct investigations in geographically diverse seronegative populations whose viral subtype and route of exposure differ: 1) MSM from the Seattle ES cohort, exposed to subtype B HIV-1; 2) high HIV risk MSM participants of HPTN Protocol 039 in Seattle and Peru, exposed to subtype B HIV-1, and 3) high risk heterosexual women and men from three cohort studies in cDurban, Republic of South Africa, exposed to subtype C HIV-1. The results will inform our decisions of the specific assays to employ in vaccine trials, guide interpretation of responses in trial participants with high-risk activities in HIV endemic areas, and potentially invoke new concepts for vaccine design and correlates of protection.